Several publications are referenced in this application by numerals in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Apoptosis is a type of cell death that appears to be under direct genetic control. During apoptosis, cells lose their cell junctions and microvilli. The cytoplasm condenses and nuclear chromatin marginates into a number of discrete masses. While the nucleus fragments, the cytoplasm contracts and mitochondria and ribosomes become densely compacted. After dilation of the endoplasmic reticulum and its fusion with the plasma membrane, the cell breaks up into several membrane bound vesicles, also known as apoptotic bodies, which are usually phagocytosed by adjacent cells.
Apoptosis induced by numerous cytotoxic agents can be suppressed by expression of the gene bcl-2 which produces a cytoplasmic protein BCL2. The bcl-2 gene was initially cloned from the t(14,18) (q32,q21) breakpoint commonly found in follicular lymphomas (1-3). BCL2 protein prevents programmed cell death in a number of systems, including B-lymphocytes. Bcl-2 appears to be expressed primarily in tissues in which cells undergo apoptosis. Transgenic mice experiments suggest that bcl-2 plays a physiologic role in T-lymphocyte selection in the thymus.
Low grade non-Hodgkin's lymphomas, including most follicular lymphomas, are considered incurable with current therapy, although bone marrow transplantation may alter this outlook for selected patients. The follicular lymphomas are characterized (85-90% of cases) by the chromosomal translocation t(14,18), in which the breakpoint on chromosome 18 is in the 3' untranslated portion of the bcl-2 gene and that on chromosome 14 is in the immunoglobulin heavy chain J region. These are joined in a head-to-tail fashion and result in a fusion messenger RNA encoding a full-length BCL2 gene product with altered regulation, generally highly expressed due to the attached immunoglobulin gene. BCL2 has oncogenic properties. Transgenic mice that overexpress the bcl-2/immunoglobulin fusion gene accumulate large numbers of small B-lymphocytes and in many ways mimic low-grade lymphoma, supporting the concept that dysregulated expression of this fusion gene is important in the pathogenesis of follicular lymphoma.
Growth inhibition by down-regulation of bcl-2 with antisense oligonucleotides specific for the bcl-2 promoter region has previously been reported (4-6). However, in vivo, it is not always optimal to target oncogenic sequences for antisense therapy due to non-specific deleterious effects on normal cells. Thus, there continues to be considerable research activity relating to antisense molecules specific for sequences at chromosomal translocation breakpoints present in various malignant tumors. An important goal of such research is to inhibit aberrant cellular proliferation by targeting such breakpoints. To date, targeting the non-oncogenic sequences of fusion transcripts so as to expand the potential for tumor specific genetic manipulation has not yet been explored.